The research proposed in this application is designed to evaluate the contributions made by messenger RNA-ribosomal RNA interaction, initiator codon sequence, and mRNA secondary structure in determining the specificity of ribosome recognition and the overall efficiency of a discrete set of translation initiation sites in the E. coli lac operon gene encoding the lactose repressor protein (I gene). A sequence of 214 nucleotides at the 5' terminus of the I gene mRNA synthesized in vitro has recently been determined by the principal investigator. This portion of the messenger includes the regions which signal initiation of both the wild type repressor protein and three internal restart polypeptides whose synthesis is activated by nonsense mutations early in the I gene. In addition, the sequence reveals a number of in-phase GUGs which have not been shown previously to serve as reinitiation codons, and it contains specific residues capable of forming a very stable hairpin structure closed by a 10 base-pair stem. Experiments using the combined techniques of mRNA and DNA sequencing, ribosome binding, and N-terminal amino acid sequencing to analyze I gene mutants specifically affected in initiation are proposed, with the aim of dissecting the functional elements of the wild type initiator and of determining which I mRNA sequence and structural features underly the apparent specificity of reinitiation site selection. Conditions will be established for the isolation of specific regions of I mRNA labelled in vivo. The possibility that transcription termination within the I gene, aggravated by ribosome release at polar nonsense mutations, is an additional factor reflected in the pattern of reinitiation observed in vivo will also be investigated. The ease of fine structure genetic analysis, nucleic acid sequence determination, and identification of in vivo translational start sites provided by this experimental system should result in substantial contributions by this study to our understanding of the molecular interactions which underly and regulate this step of gene expression.